A second cauliflower mosaic virus gene product influences the structure of the viral inclusion body

We have used electron microscopy of thin sections and experiments on isolated viroplasms to compare the properties of four strains of cauliflower mosaic virus (CaMV), three of which were partially or completely deleted in open reading frame (ORF) II. Our results confirm that this gene is required for aphid transmissibility and show that the product of ORF II influences the firmness with which virions are held within the viroplasm. Analysis of the proteins in the viroplasms showed that a mutant with a partial deletion in ORF II produced a protein smaller than the normal ORF product. This smaller protein was non-functional with respect both to aphid transmissibility and properties of the viroplasms.     

Isolation and characterization of faithful and altered clones of the genomes of cauliflower mosaic virus isolates Cabb B-JI, CM4-184, and Bari I

Full-length genomes of cauliflower mosaic virus (CaMV) isolates Cabb B-JI, CM4-184, and Bari I have been cloned in the SalGI site of plasmid pAT 153. The cloned DNAs were characterized by restriction mapping and infectivity assays. All the sites present in the virion DNAs were found in the cloned DNAs. Comparison of restriction maps with those of DNA from two other isolates which have been recently completely sequenced revealed a close relationship among the different isolates. Some of the clones appear to be faithful copies of the viral genomes and these viral inserts are infectious when inoculated into turnip plants. Various clones with deletions in the CaMV DNA have been isolated and characterized. Some of them may correspond to deletions naturally occurring in a subpopulation of the virus whereas others occurred during cloning. None of the deleted fragments are infectious when inoculated into plants. Strikingly, all the deletions overlap one or two of the specific single-stranded breaks characteristic of caulimoviruses, suggesting that sequences surrounding the breaks are not dispensable.     

Aphid transmission of cauliflower mosaic virus requires the viral PIII protein

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids.     

Pathogenic interactions between variants of cauliflower mosaic virus and Arabidopsis thaliana

Pathogenic interactions between genetic variants of cauliflower mosaic virus (CaMV) and Arabidopsis thaliana were characterized to identify combinations potentially useful in molecular genetic analysis. Infections of a glabrous mutant (gl1) of Arabidopsis ecotype Columbia (Col-0 gl1) by 30 CaMV isolates were assessed by recording symptom character. Thirteen isolates failed to cause symptoms; the remainder induced symptoms that varied between mild and very severe. Some CaMV isolates produced symptoms in Arabidopsis that differed significantly in severity or character from those produced in a standard host Brassica rapa (turnip). A greater variety of symptom types was observed in a single Arabidopsis ecotype infected with a range of CaMV isolates than was found in a range of Arabidopsis ecotypes infected with a single, typical CaMV isolate (Cabb B-JI). One isolate, Bari-1, that was asymptomatic but accumulated virus in Arabidopsis ecotype Col-0 gl1, caused mild symptoms in ecotype Ler gl1. A hybrid virus constructed from CaMV isolates Cabb B-JI and Bari-1 produced symptoms in Arabidopsis variants that were more severe than in either parental isolate. From a screen of EMS-mutagenized Arabidopsis, one mutant (Col-0 dv1) with a pale-green, dark-vein phenotype which had an altered symptom response to CaMV, was isolated. From this, a phenotypically near-normal revertant (Col-0 dv1R) spontaneously arose, but which showed altered responses to CaMV. Infection of Col-0 dv1R by CaMV isolate Bari-1 elicited symptoms unlike the parent Arabidopsis ecotype (Col-0 gl1). Also, Col-0 dv1 and Col-0 dv1R expressed an uncharacteristic necrotic reaction to CaMV.     

The cauliflower mosaic virus virion-associated protein is dispensable for viral replication in single cells

Cauliflower mosaic virus (CaMV) open reading frame III (ORF III) codes for a virion-associated protein (Vap), which is one of two viral proteins essential for aphid transmission. However, unlike the aphid transmission factor encoded by CaMV ORF II, Vap is also essential for systemic infection, suggesting that it is a multifunctional protein. To elucidate the additional function or functions of Vap, we tested the replication of noninfectious ORF III-defective mutants in transfected turnip protoplasts. PCR and Western blot analyses revealed that CaMV replication had occurred with an efficiency similar to that of wild-type virus and without leading to reversions. Electron microscopic examination revealed that an ORF III frameshift mutant formed normally structured virions. These results demonstrate that Vap is dispensable for replication in single cells and is not essential for virion morphogenesis. Analysis of inoculated turnip leaves showed that the ORF III frameshift mutant does not cause any detectable local infection. These results are strongly indicative of a role for Vap in virus movement.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

Transmissibility of Broad bean wilt virus 1 by aphids: influence of virus accumulation in plants,virus genotype and aphid species

Broad bean wilt virus 1 (BBWV‐1) is transmitted by several aphid species in a non‐persistent manner. Transmission efficiency by vectors is a key factor for understanding virus epidemiology and applying disease control measures based on limiting virus spread. We evaluated the transmission rates of two genetically divergent BBWV‐1 isolates (PV‐132 from USA and Ben from Spain) infecting broad bean (Vicia faba L.) by isofemale lines of nine aphid species from eight different genera collected in Spain. Our analyses showed that: (a) the virus concentration in the source plant was a key factor in BBWV‐1 transmissibility; (b) The Spanish isolate Ben was transmitted more efficiently than the American isolate PV‐132 by most aphid species, but this was only due to the higher accumulation of Ben in plants, as both isolates had similar transmissibility after adjusting virus concentration and (c) The transmission rate varied greatly between the different aphid species.     

Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

Comparison of short- and long-feed transmission of the cauliflower mosaic virus Cabb-S strain and SΔII hybrid by two species of aphid: Myzus persicae (Sulzer) and Brevicoryne brassicae (L.)

The cauliflower mosaic virus (CaMV) hybrid SΔII, partially deleted in ORFII, loses its transmissibility by the aphid Myzus persicae on 5-min acquisition feed. We have also shown that it is not transmitted after 8-h acquisition feed. The same occurs with Brevicoryne brassicae. Therefore, the aphid transmission factor (ATF) is involved in both means of transmission and in both aphid species. M. persicae can acquire CaMV Cabb-S strain in less than 20 s. M. persicae is a more efficient vector during a short feed than during a long feed, contrary to B. brassicae which transmits better during a long feed.     

Solubilization of the membrane proteins from Semliki Forest virus with Triton X100

Increasing concentrations of Triton X100 have been found to cause stepwise dissociation of the membrane of Semliki Forest virus. The final stage of the breakdown process leads to solubilization of the membrane proteins which can be separated from the membrane lipids and the viral nucleocapsid by density gradient centrifugation in the presence of 0.05% Triton X100. Two different forms of Semliki Forest virus protein have been observed with sedimentation coefficients of approximately 4 S and 23 S. The 4 S aggregate appears to consist of two polypeptide chains complexed with about 75 molecules of Triton X100. The 23 S form is a rosette-like aggregate containing about 16 polypeptide chains and about 260 molecules of Triton X100. Sucrose alters the equilibrium between the 4 S and 23 S forms: removal of sucrose leads to association of the 4 S form to the 23 S form and addition of sucrose to dissociation.A scheme for the dissociation of the Semliki Forest virus membrane is presented which is discussed with reference to other biological membranes. It is suggested that Triton X100 and deoxycholate solubilize amphipathic membrane proteins by binding to the hydrophobic segments of these proteins.     

A Distinct Subpopulation of Cauliflower mosaic virus (CaMV) in South Iran

Cauliflower mosaic virus (CaMV) with a high incidence and widespread distribution on Brassica crops in Iran reduces the yield and quality of these crops. The complete sequences of three open reading frames (ORFs) 2, 4 and 6 coding for aphid transmission (AT), coat protein (CP) and inclusion body protein/translation transactivator (TAV) genes, respectively, were determined for two Iranian CaMV isolates from Kerman (south Iran). They induced latent or mild mottle (L/MMo) infection in Brassica oleracea var. capitata so are considered as the (L/MMo) biotype. Clear recombination breakpoints were detected between ORF2 and ORF6 in two Kerman isolates using concatenate fragments. Phylogenetic analysis revealed three Iranian CaMV subpopulations in which the two Kerman isolates in the new subgroup C were added to the two previously reported Iranian subpopulations A (central and west Iran) and B (north‐east Iran). Also three regions of pairwise identity were detected which representing: 97.1–100, 93.8–97.1 and 90.6–93.8% for subgroups A, C and B, respectively. Our analysis showed the high variability of Iranian CaMV population and provided valuable new information for understanding the diversity and evolution of caulimoviruses. Furthermore, star phylogeny was found in the subgroup C with overall lack of nt diversity and high haplotype diversity as evidence of a recent population expansion after a genetic bottleneck although this may have been modified subsequently by clinal genetic drift. The appearance of new genetic types demonstrates a high potential of risks and should be considered in the planning of efficient control programmes.     

Cauliflower mosaic virus protein P6-TAV plays a major role in alteration of aphid vector feeding behaviour but not performance on infected Arabidopsis

Emerging evidence suggests that viral infection modifies host plant traits that in turn alter behaviour and performance of vectors colonizing the plants in a way conducive for transmission of both nonpersistent and persistent viruses. Similar evidence for semipersistent viruses like cauliflower mosaic virus (CaMV) is scarce. Here we compared the effects of Arabidopsis infection with mild (CM) and severe (JI) CaMV isolates on the feeding behaviour (recorded by the electrical penetration graph technique) and fecundity of the aphid vector Myzus persicae. Compared to mock-inoculated plants, feeding behaviour was altered similarly on CM- and JI-infected plants, but only aphids on JI-infected plants had reduced fecundity. To evaluate the role of the multifunctional CaMV protein P6-TAV, aphid feeding behaviour and fecundity were tested on transgenic Arabidopsis plants expressing wild-type (wt) and mutant versions of P6-TAV. In contrast to viral infection, aphid fecundity was unchanged on all transgenic lines, suggesting that other viral factors compromise fecundity. Aphid feeding behaviour was modified on wt P6-CM-, but not on wt P6-JI-expressing plants. Analysis of plants expressing P6 mutants identified N-terminal P6 domains contributing to modification of feeding behaviour. Taken together, we show that CaMV infection can modify both aphid fecundity and feeding behaviour and that P6 is only involved in the latter.     

Solubilization of the semliki forest virus membrane with sodium deoxycholate

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest virus have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at $\text{0.9 ± 0.1}\text{mM}$ free equilibrium concentration when $\text{2.2 ± 0.2 ß 10}{}^3\text{mol}$ of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at $\text{1.5 ± 0.1}\text{mM}$ sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.